STRATEGIES TO SUPPRESS AGGREGATION OF RECOMBINANT KERATINOCYTE GROWTH-FACTOR DURING LIQUID FORMULATION DEVELOPMENT

Recombinant human keratinocyte growth factor (rhKGF) is a fairly unsta ble protein, posing a challenging problem for long-term storage. Durin g storage, the protein unfolds at relatively low temperatures and the unfolded proteins aggregate rapidly, leading to the formation of large visible precipitates. Thermal unfolding of rhKGF displays a similar p attern, i.e., unfolding is followed immediately by aggregation as the temperature is increased. As the unfolding and aggregation (precipitat ion) of rhKGF limit the storage life of the protein, a search for stab ilizers to suppress rhKGF unfolding and aggregation has been done by e xamining the effects of excipients on thermal melting temperature and on the rate of protein aggregation during storage. Sulfated polysaccha rides and citrate are found to be effective in increasing the melting temperature of rhKGF or preventing its aggregation. In particular, 0.5 % (w/v) heparin and high molecular weight dextran sulfate, and 0.5 M c itrate are highly effective, decreasing the rates of rhKGF aggregation by about 50-fold. Other negatively charged small ions, such as phosph ate, also have moderate stabilizing effects on rhKGF. A mechanistic st udy of the aggregation pathway of rhKGF has led to a better understand ing of the stabilizing effects of these molecules. Molecules which enh ance rhKGF conformational stability are capable of effectively suppres sing rhKGF aggregation.